Qtrap

MCF-7 cells and MCF7-GPD1 cells were harvested by trypsinization, rinsed twice in ice-cold PBS by low-speed centrifugation at 4 °C, and stored at −80 °C until analysis. The samples were extracted by the extraction solvent (methyl tert-butyl ether: methanol = 3:1, v/v) containing internal standard mixture on ice for 15 min and added into 200 μL water to centrifuge at 12,000 rpm for 10 min. 200 μL of the upper organic layer was collected and evaporated using a vacuum concentrator. The dry extract was reconstituted using 200 μL mobile phase B before LC-MS/MS analysis [10 (link)].
The full details of the lipidomics methods were explained in the Supplementary Material. In brief, the sample extracts were analyzed using ultra-performance liquid chromatography (UPLC)-ESI-MS/MS system (UPLC: ExionLC AD; MS: QTRAP, Sciex, USA). Differential metabolites (DMs) were identified in the Lipid Maps databases as the threshold of VIP (variable importance in projection) ≥ 1, p-value <0.05, and absolute log₂FC (fold change) ≥ 1. Pathway enrichment analysis and function annotations of metabolites were performed using the Kyoto Encyclopedia of Genes and Genomes (KEGG) and HMDB database [10 (link)]. The experiment was completed by METWARE Inc (Wuhan, China).

The LIT and triple quadrupole (QQQ) scans were obtained using a triple quadrupole-linear ion trap mass spectrometer (Q TRAP) (Sciex, Framingham, MA, USA), API 6500 Q TRAP LC/MS/MS System (Sciex), equipped with an ESI Turbo Ion-Spray interface (Sciex), operating in a positive ion mode and controlled using the analyst 1.6.3 software (Sciex). The ESI source operation parameters were as follows: ion source, turbo spray; source temperature, 500 °C; ion spray voltage, (IS) 5500 V; ion source gas I (GSI), gas II (GSII), and curtain gas (CUR) were set at 55, 60, and 25.0 psi, respectively; the collision gas (CAD) was high. Instrument tuning and mass calibration were performed with 10 and 100 μmol/L polypropylene glycol solutions in QQQ and LIT modes. QQQ scans were acquired as multiple reaction monitoring (MRM) experiments with collision gas (nitrogen) set at 5 psi. The declustering potential (DP) and collision energy (CE) for individual MRM transitions were done with further DP and CE optimization. A specific set of MRM transitions were monitored for each period according to the metabolites eluted within the period.

The data acquisition instrumentation system mainly comprises Ultra Performance Liquid Chromatography (UPLC) (EXionLC AD, https://sciex.com.cn/, accessed on 1 September 2021) and Tandem Mass Spectrometry (MS/MS) (QTRAP^®, https://sciex.com.cn/, accessed on 1 September 2021). The chromatographic column was a Waters ACQUI T YUPLCHSS 13CI8 (1.8 µm, 2.1 mm*100 mm). The mobile phase was ultra-pure water (0.1% formic acid) in the A phase and pure acetonitrile (0.1% formic acid) in the B phase. The elution gradient was 0 min water/acetonitrile (95:5 v/v), 11 min, 10:90 v/v, 12.0 min, 10.90 v/v, 12 min, 95:5 v/v, 14.0 min, 95.5 v/v. The separation conditions were set as follows: flow rate 0.4 mL/min, column temperature 40 °C, injection volume 5 uL.
The mass acquisition conditions were as follows: electrospray ionization (ESI) at 500 °C, ion spray voltage (IS) 5500 V (positive), −4500 V (negative), ion source gas I (GSI) at 55 psi, gas II (GSII) at 60 psi, curtain gas (CUR) at 25 psi, and the collision-activated dissociation (CAD) parameter was set to high. In the triple quadrupole (QTRAP), each ion pair was scanned for detection according to the optimized declustering potential (DP) and collision energy (CE).

Ultra-performance liquid chromatography (UPLC) (Shim-pack UFLC SHIMADZU CBM30A, https://www.shimadzu.com/) and tandem mass spectrometry (QTRAP^®, https://sciex.com/) were used to analyze the chromatograms and mass spectra of the sample extracts. The liquid phase conditions were listed as follows: UPLC: column, Waters ACQUITY UPLC HSS T3 C18 (1.8 µm, 2.1 mm × 100 mm); solvent system, ultrapure water (0.04% acetic acid): acetonitrile (0.04% acetic acid); gradient program, 95:5 V/V at 0 min, 5:95 V/V at 11.0 min, 5:95 V/V at 12.0 min, 95:5 V/V at 12.1 min, 95:5 V/V at 14.0 min; flow rate, 0.4 mL/min; column temperature, 40 °C; injection volume 2 μL [15 (link)].
Mass spectrometry operation parameters were as follows: electrospray ionization (ESI) temperature 500 °C; mass spectrometry voltage 5500 V (positive), −4500 V (negative); ion source gas I (GSI), gas II (GSII), curtain gas (CUR) were set at 55, 60, 25 psi, respectively; the collision-activated dissociation (CAD) was set to high. Each ion-pair was scanned according to the optimized declustering potential (DP) and collision energy (CE) in the triple quadrupole (Q trap) [16 (link)].

The mass spectrometry analysis was performed on a Lipidyzer™ Platform, including the Sciex QTRAP® (SCIEX, Darmstadt, Germany) 4500 system. Multiple reaction monitoring (MRM) was used to target and quantitate several hundreds of lipids molecular. All samples were first measured in positive and negative polarity with SelexION separation, followed by measurement without SelexION separation. Lipids were identified based on their retention time, and mass spectral information. We provide this information (filename-identification method.xlsx). All data were acquired and processed automatically using the Lipid Manager Workflow software (SCIEX, Darmstadt, Germany).